Since establishment in human of embryonic stem cells (ES cells) retaining pluripotency of being capable of differentiating into all cells in an individual and simultaneously having self-replication potential of being capable of infinitely proliferating, studies to induce in vitro differentiation of ES cells into principal body part cells are actively carried out.
Since production of human induced pluripotent stem cells (iPS cells) having pluripotency and self-replication potential together such as ES cells by introducing a specific gene into a somatic cell, studies to induce in vitro differentiation into principal body part cells using iPS cells in addition to ES cells are actively carried out.
For example, it is known that mouse ES cells can maintain an undifferentiating property by co-culturing on feeder cells such as mouse embryonic fibroblasts (MEF) and the like in the presence of a leukemia inhibitory factor (LIF) and if LIF is removed from a maintenance culture medium, differentiation of ES cells is induced (non-patent document 1).
On the other hand, it is reported that also human ES cells and human iPS cells can maintain undifferentiation by co-culturing on feeder cells such MEF and the like, and differentiation thereof is induced by transferring to a differentiation culture medium (non-patent document 2).
Further, it is reported that since a threonine dehydrogenase gene is expressed remarkably in mouse ES cells, proliferation of the cells depends on the metabolism of threonine as one of amino acids (non-patent document 3).
ES cells and iPS cells are useful models for study of gene functions in the development stage and possibly become medical transplantable cell sources because of pluripotency thereof, therefore, application of ES cells and iPS cells to regenerative medicine is expected. However, control of differentiation is necessary for use of ES cells and iPS cells in cell replacement therapies and tissue transplantation in diseases such as, for example, diabetes mellitus and the like.
Then, studies to control differentiation from undifferentiated cells into tissue cells are actively conducted recently. For example, there is a report on in vitro production of endoderm cells and insulin-producing cells from mouse and human ES cells (non-patent document 4, non-patent document 5).
Further, there are reports on a method of efficiently inducing differentiation of ES cells into pancreatic precursor cells by using mouse mesonephric cell line M15 cells as a supporting cell and adding activin⋅FGF (fibroblast growth factor)⋅retinoic acid to the culture medium (patent document 1, non-patent document 6) and on a method of efficiently inducing differentiation of mouse and human ES cells into hepatocytes, byway of culture conditions in which mmcM15 cells are used as a supporting cell and specific secreted growth factors (FGF and BMP (Bone Morphogenetic Proteins)) are added or eliminated (patent document 2, non-patent document 7).
When differentiation of ES cells and iPS cells into various cells is induced, however, undifferentiated stem cells partially remain and mix. In regenerative medicine, there is fear regarding safeness such as a possibility of canceration of such cells. For this reason, there is a need for a technology of eliminating undifferentiated cells in differentiated cells or of preventing mixing of undifferentiated cells by increasing differentiation induction efficiency.
There are various methods suggested for confirming mixing of undifferentiated cells or for selection thereof.
For example, there is a method of eliminating undifferentiated ES cells remaining after induction of differentiation, by utilizing a promoter of Stmt as an undifferentiation specific marker, thereby facilitating selection of undifferentiated ES cells remaining after induction of differentiation, and by removing the undifferentiated ES cells (patent document 3).
Further, there is a method of eliminating undifferentiated human embryonic stem cells in which undifferentiated human embryonic stem cells are identified by identifying cells expressing on its surface a podocalyxin like protein, and these cells are isolated (patent document 4).
It has recently reported that human ES cells and human iPS cells show a remarkable difference in differentiation potential between cell lines and, a tendency of differentiating into a specific family, that is, differentiation directionality varies for every cell line (non-patent document 8).
It is believed that banking of patient-derived iPS cell lines progresses in the field of regenerative medicine and it is expected that differentiation of many kinds of iPS cell lines into desired cells is induced, in the days ahead. It is supposed that if differentiation resistance is present between lines due to differentiation directionality for every line, when differentiation is induced under the same condition, differentiation induction efficiency lowers and undifferentiated cells mix in large amount and the like, affecting the nature of the final product. Accordingly, a method capable of efficiently inducing differentiation into intended tissue while avoiding differentiation resistance between lines is desired.